RESUMEN

African swine fever virus causes a lethal hemorrhagic disease in domestic swine and wild boar for which currently licensed commercial vaccines are only available in Vietnam. Development of subunit vaccines is complicated by the lack of information on protective antigens as well as suitable delivery systems. Our previous work showed that a pool of eight African swine fever virus genes vectored using an adenovirus prime and modified vaccinia virus boost could prevent fatal disease after challenge with a virulent genotype I isolate of the virus. Here, we identify antigens within this pool of eight that are essential for the observed protection and demonstrate that adenovirus-prime followed by adenovirus-boost can also induce protective immune responses against genotype I African swine fever virus. Immunization with a pool of adenoviruses expressing individual African swine fever virus genes partially tailored to genotype II virus did not protect against challenge with genotype II Georgia 2007/1 strain, suggesting that different antigens may be required to induce cross-protection for genetically distinct viruses. IMPORTANCE: African swine fever virus causes a lethal hemorrhagic disease in domestic pigs and has killed millions of animals across Europe and Asia since 2007. Development of safe and effective subunit vaccines against African swine fever has been problematic due to the complexity of the virus and a poor understanding of protective immunity. In a previous study, we demonstrated that a complex combination of eight different virus genes delivered using two different viral vector vaccine platforms protected domestic pigs from fatal disease. In this study, we show that three of the eight genes are required for protection and that one viral vector is sufficient, significantly reducing the complexity of the vaccine. Unfortunately, this combination did not protect against the current outbreak strain of African swine fever virus, suggesting that more work to identify immunogenic and protective viral proteins is required to develop a truly effective African swine fever vaccine.

Asunto(s)

Adenoviridae , Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Vectores Genéticos , Genotipo , Vacunas Virales , Animales , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/inmunología , Fiebre Porcina Africana/prevención & control , Fiebre Porcina Africana/virología , Fiebre Porcina Africana/inmunología , Porcinos , Vacunas Virales/inmunología , Vacunas Virales/genética , Vacunas Virales/administración & dosificación , Vectores Genéticos/genética , Adenoviridae/genética , Adenoviridae/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/genética , Antígenos Virales/inmunología , Antígenos Virales/genética

RESUMEN

Bispecific T cell engagers are a promising class of therapeutic proteins for cancer therapy. Their potency and small size often come with systemic toxicity and short half-life, making intravenous administration cumbersome. These limitations can be overcome by tumor-specific in situ expression, allowing high local accumulation while reducing systemic concentrations. However, encoding T cell engagers in viral or non-viral vectors and expressing them in situ ablates all forms of quality control performed during recombinant protein production. It is therefore vital to design constructs that feature minimal domain mispairing, and increased homogeneity of the therapeutic product. Here, we report a T cell engager architecture specifically designed for vector-mediated immunotherapy. It is based on a fusion of a designed ankyrin repeat protein (DARPin) to a CD3-targeting single-chain antibody fragment, termed DATE (DARPin-fused T cell Engager). The DATE induces potent T cell-mediated killing of HER2+ cancer cells, both as recombinantly produced therapeutic protein and as in situ expressed payload from a HER2+-retargeted high-capacity adenoviral vector (HC-AdV). We report remarkable tumor remission, DATE accumulation, and T cell infiltration through in situ expression mediated by a HER2+-retargeted HC-AdV in vivo. Our results support further investigations and developments of DATEs as payloads for vector-mediated immunotherapy.

RESUMEN

Neoantigen (neoAg)-based cancer vaccines expand preexisting antitumor immunity and elicit novel cancer-specific T cells. However, at odds with prophylactic vaccines, therapeutic antitumor immunity must be induced when the tumor is present and has already established an immunosuppressive environment capable of rapidly impairing the function of anticancer neoAg T cells, thereby leading to lack of efficacy. To overcome tumor-induced immunosuppression, we first vaccinated mice bearing immune checkpoint inhibitor (CPI)-resistant tumors with an adenovirus vector encoding a set of potent cancer-exogenous CD8 and CD4 T cell epitopes (Ad-CAP1), and then "taught" cancer cells to express the same epitopes by using a tumor-retargeted herpesvirus vector (THV-CAP1). Potent CD8 effector T lymphocytes were elicited by Ad-CAP1, and subsequent THV-CAP1 delivery led to a significant delay in tumor growth and even cure.

RESUMEN

Delivering vectorized information into cells with the help of viruses has been of high interest to fundamental and applied science, and bears significant therapeutic promise. Human adenoviruses (HAdVs) have been at the forefront of gene delivery for many years, and the subject of intensive development resulting in several generations of agents, including replication-competent, -defective or retargeted vectors, and recently also helper-dependent (HD), so-called gutless vectors lacking any viral protein coding information. While it is possible to produce HD-AdVs in significant amounts, physical properties of these virus-like particles and their efficiency of transduction have not been addressed. Here, we used single-cell and single virus particle assays to probe the effect of genome length on HAdV-C5 vector transduction. Our results demonstrate that first-generation C5 vectors lacking the E1/E3 regions of the viral genome as well as HD-AdV-C5 particles with a wild type (wt) ∼36 kbp or an undersized double-strand DNA genome are similar to human adenovirus C5 (HAdV-C5) wt regarding attachment to human lung epithelial cells, endocytic uptake, endosome penetration and dependency on the E3 RING ubiquitin ligase Mind Bomb 1 for DNA uncoating at the nuclear pore complex. Atomic force microscopy measurements of single virus particles indicated that small changes in the genome length from 94% to 103% of HAdV-C5 have no major impact on physical and mechanical features of AdV vectors. In contrast, an HD-AdV-C5 with ∼30 kbp genome was slightly stiffer and less heat-resistant than the other particles, despite comparable entry and transduction efficiencies in tissue culture cell lines, including murine alveolar macrophage-like Max-Planck-Institute (MPI)-2 cells. Together, our in vitro studies reinforce the use of HD-AdV vectors for effective single round gene delivery. The results illustrate how physical properties and cell entry behavior of single virus particles can provide functional information for anticipated therapeutic vector applications.

Asunto(s)

Adenoviridae , Adenovirus Humanos , Animales , Humanos , Ratones , Adenoviridae/genética , Adenovirus Humanos/genética , Línea Celular , Vectores Genéticos , ADN

RESUMEN

BACKGROUND: Hepatitis C virus (HCV) vaccines are an urgent need to prevent hepatitis C and its further progression of hepatocellular carcinoma. Since the promising T cell based chimpanzee adenovirus and modified vaccinia virus Ankara vectorial HCV vaccines were failed in clinical phase II trial, the vaccine designs to improve protection efficacy in combination of cellular and humoral immunity have been hypothesized against multi-genotypic HCV. METHODS: Eight HCV vaccine strains were constructed with two novel adenovirus vectors (Sad23L and Ad49L) encoding E1E2 or NS3-5B proteins of HCV genotype (Gt) 1b and 6a isolates, covering 80 % HCV strains prevalent in south China and south-east Asia. Eight HCV vaccine strains were grouped into Sad23L-based vaccine cocktail-1 and Ad49L-based vaccine cocktail-2 for vaccinating mice, respectively. RESULTS: The immunogenicity of a single dose of 107-1010 PFU HCV individual vaccines was evaluated in mice, showing weak specific antibody to E1 and E2 protein but a dose-dependent T cell response to E1E2/NS3-5B peptides, which could be significantly enhanced by boosting with an alternative vector vaccine carrying homologous antigen. Prime-boost vaccinations with vaccine cocktail-1 and cocktail-2 induced significantly higher cross-reactive antibody and stronger T cell responses to HCV Gt-1b/6a. The high frequency of intrasplenic and intrahepatic NS31629-1637 CD8+ T cell responses were identified, in which the high proportion of TRM and TEM cells might play an important role against HCV infection in liver. CONCLUSIONS: Prime-boost regimens with HCV vaccine cocktails elicited the broad cross-reactive antibody and robust T cell responses against multi-genotypic HCV in mice.

Asunto(s)

Hepatitis C , Vacunas , Animales , Ratones , Hepacivirus/genética , Hepatitis C/prevención & control , Vectores Genéticos , Virus Vaccinia/genética , Adenoviridae/genética , Inmunidad , Genotipo

RESUMEN

Adenovirus vectors possess a good safety profile, an extensive genome, a range of host cells, high viral yield, and the ability to elicit broad humoral and cellular immune responses. Adenovirus vectors are widely used in infectious disease research for future vaccine development and gene therapy. In this study, we obtained a fowl adenovirus serotype 4 (FAdV-4) isolate from sick chickens with hepatitis-hydropericardium syndrome (HHS) and conducted animal regression text to clarify biological pathology. We amplified the transfer vector and extracted viral genomic DNA from infected LMH cells, then recombined the mixtures via the Gibson assembly method in vitro and electroporated them into EZ10 competent cells to construct the FAdV-4 infectious clone. The infectious clones were successfully rescued in LMH cells within 15 days of transfection. The typical cytopathic effect (CPE) and propagation titer of FAdV-4 infectious clones were also similar to those for wild-type FAdV-4. To further construct the single-cycle adenovirus (SC-Ad) vector, we constructed SC-Ad vectors by deleting the gene for IIIa capsid cement protein. The FAdV4 infectious clone vector was introduced into the ccdB cm expression cassette to replace the IIIa gene using a λ-red homologous recombination technique, and then the ccdB cm expression cassette was excised by PmeI digestion and self-ligation to obtain the resulting plasmids as SC-Ad vectors.

Asunto(s)

Enfermedades Transmisibles , Hepatitis A , Animales , Pollos , Serogrupo , Adenoviridae/genética , Proteínas de la Cápside , ADN Viral

RESUMEN

Fibroblast activation protein (FAP) is a cell surface serine protease that is highly expressed on reactive stromal fibroblasts, such as cancer-associated fibroblasts (CAFs), and generally absent in healthy adult tissues. FAP expression in the tumor stroma has been detected in more than 90% of all carcinomas, rendering CAFs excellent target cells for a tumor site-specific adenoviral delivery of cancer therapeutics. Here, we present a tropism-modified human adenovirus 5 (Ad5) vector that targets FAP through trivalent, designed ankyrin repeat protein-based retargeting adapters. We describe the development and validation of these adapters via cell-based screening assays and demonstrate adapter-mediated Ad5 retargeting to FAP+ fibroblasts in vitro and in vivo. We further show efficient in vivo delivery and in situ production of a therapeutic payload by CAFs in the tumor microenvironment (TME), resulting in attenuated tumor growth. We thus propose using our FAP-Ad5 vector to convert CAFs into a "biofactory," secreting encoded cancer therapeutics into the TME to enable a safe and effective cancer treatment.

RESUMEN

We aim to develop an in vivo hematopoietic stem cell (HSC) gene therapy approach for persistent control/protection of HIV-1 infection based on the stable expression of a secreted decoy protein for HIV receptors CD4 and CCR5 (eCD4-Ig) from blood cells. HSCs in mice and a rhesus macaque were mobilized from the bone marrow and transduced by an intravenous injection of HSC-tropic, integrating HDAd5/35++ vectors expressing rhesus eCD4-Ig. In vivo HSC transduction/selection resulted in stable serum eCD4-Ig levels of ∼100 µg/mL (mice) and >20 µg/mL (rhesus) with half maximal inhibitory concentrations (IC50s) of 1 µg/mL measured by an HIV neutralization assay. After simian-human-immunodeficiency virus D (SHIV.D) challenge of rhesus macaques injected with HDAd-eCD4-Ig or a control HDAd5/35++ vector, peak plasma viral load levels were ∼50-fold lower in the eCD4-Ig-expressing animal. Furthermore, the viral load was lower in tissues with the highest eCD4-Ig expression, specifically the spleen and lymph nodes. SHIV.D challenge triggered a selective expansion of transduced CD4+CCR5+ cells, thereby increasing serum eCD4-Ig levels. The latter, however, broke immune tolerance and triggered anti-eCD4-Ig antibody responses, which could have contributed to the inability to eliminate SHIV.D. Our data will guide us in the improvement of the in vivo approach. Clearly, our conclusions need to be validated in larger animal cohorts.

Asunto(s)

Infecciones por VIH , VIH-1 , Síndrome de Inmunodeficiencia Adquirida del Simio , Virus de la Inmunodeficiencia de los Simios , Humanos , Animales , Ratones , Macaca mulatta , Virus de la Inmunodeficiencia de los Simios/genética , Células Madre Hematopoyéticas , Síndrome de Inmunodeficiencia Adquirida del Simio/terapia

RESUMEN

The consequences of human adenovirus (HAdV) infections are generally mild. However, despite the perception that HAdVs are harmless, infections can cause severe disease in certain individuals, including newborns, the immunocompromised, and those with pre-existing conditions, including respiratory or cardiac disease. In addition, HAdV outbreaks remain relatively common events and the recent emergence of more pathogenic genomic variants of various genotypes has been well documented. Coupled with evidence of zoonotic transmission, interspecies recombination, and the lack of approved AdV antivirals or widely available vaccines, HAdVs remain a threat to public health. At the same time, the detailed understanding of AdV biology garnered over nearly 7 decades of study has made this group of viruses a molecular workhorse for vaccine and gene therapy applications.

Asunto(s)

Infecciones por Adenovirus Humanos , Adenovirus Humanos , Infecciones del Sistema Respiratorio , Recién Nacido , Humanos , Adenoviridae/genética , Infecciones por Adenovirus Humanos/epidemiología , Adenovirus Humanos/genética , Genómica , Genotipo , Filogenia

RESUMEN

Since the isolation of adenovirus (AdV) in 1953, AdVs have been used as vectors for various therapeutic purposes, such as gene therapy in cancers and other malignancies, vaccine development and delivery of CRISPR-Cas9 machinery. Over the years, several AdV vector modifications have been introduced, including fiber switching, incorporation of ligands in the viral capsid and hexon modification of the fiber, to improve the efficiency of AdV as a vector. CRISPR-Cas9 has recently been used for these modifications and is also used in other adeno-associated viruses. These modifications further allow the production of AdV libraries that display random peptides for the production of cancer-targeting AdV vectors. This review focuses on the common methods of AdV construction, changes in AdV tropism for the improvement of therapeutic efficiency and the role of AdV vectors in gene therapy, vaccine development and CRISPR-Cas9 delivery.

Asunto(s)

Adenoviridae , Vectores Genéticos , Vectores Genéticos/genética , Adenoviridae/genética , Terapia Genética

RESUMEN

BACKGROUND: Respiratory syncytial virus (RSV) remains a leading cause of pediatric morbidity, with no approved vaccine. We assessed the safety and immunogenicity of the Ad26.RSV.preF vaccine candidate in adults and children. METHODS: In this randomized, double-blind, phase 1/2a, placebo-controlled study, 12 adults (18-50 years) and 36 RSV-seropositive children (12-24 months) were randomized 2:1 to Ad26.RSV.preF (1 × 1011 viral particles [vp] for adults, 5 × 1010 vp for children) or placebo, at day 1 and 29, with 6-month immunogenicity and 1-year safety follow-up. Respiratory syncytial virus infection was an exploratory outcome in children. RESULTS: In adults, solicited adverse events (AEs) were generally mild to moderate, with no serious AEs. In children, no vaccination-related serious AEs were reported; fever was reported in 14 (58.3%) Ad26.RSV.preF recipients. Baseline pediatric geometric mean titers for RSV A2 neutralization increased from 121 (95% confidence interval [CI], 76-191) to 1608 (95% CI, 730-3544) at day 29, and 2235 (95% CI, 1586-3150) at day 57, remaining elevated over 7 months. Respiratory syncytial virus infection was confirmed in fewer children receiving Ad26.RSV.preF (1, 4.2%) than placebo (5, 41.7%). CONCLUSIONS: Ad26.RSV.preF demonstrated immunogenicity in healthy adults and toddlers, with no safety concerns raised. Evaluations in RSV-seronegative children are underway.

Asunto(s)

Infecciones por Virus Sincitial Respiratorio , Vacunas contra Virus Sincitial Respiratorio , Virus Sincitial Respiratorio Humano , Humanos , Adulto , Niño , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Virus Sincitial Respiratorio Humano/genética , Adenoviridae/genética , Inmunogenicidad Vacunal

RESUMEN

Adenovirus vectors offer a convenient platform for the expression of antigens and have become an attractive system for vaccine development. Currently, the most successful approach to the development of new foot-and-mouth disease (FMD) vaccines has been the production of a replication-defective human serotype 5 adenovirus that delivers the capsid and capsid processing coding regions of FMD virus (FMDV) (Ad5-FMD). A specific construct for FMDV serotype A24 has been fully developed into a commercial product fulfilling the requirements of the Center of Veterinary Biologics (CVB) of the Animal and Plant Health Inspection Service (APHIS) of the U.S. Department of Agriculture (USDA), for commercialization in the USA. In this chapter, we describe a standard protocol for the generation and small-scale production of Ad5-FMDV serotype O1Manisa vaccines. We use directional cloning to introduce the FMDV O1Manisa capsid in the Ad5-Blue vector. This is followed by the linearization of the recombinant Ad5 with Pac I and transfection into HEK293 cells for rescue and propagation, and then by increased production and purification. Finally, purified recombinant virus is characterized by determining virus yield and expression of targeted antigen in specific cell type of interest.

Asunto(s)

Adenovirus Humanos , Virus de la Fiebre Aftosa , Fiebre Aftosa , Vacunas Virales , Adenovirus Humanos/genética , Animales , Anticuerpos Antivirales , Fiebre Aftosa/prevención & control , Virus de la Fiebre Aftosa/genética , Vectores Genéticos/genética , Células HEK293 , Humanos , Vacunas Sintéticas , Vacunas Virales/genética

RESUMEN

BACKGROUND: Despite the high disease burden of respiratory syncytial virus (RSV) in older adults, there is no approved vaccine. We evaluated the experimental RSV vaccine, Ad26.RSV.preF, a replication-incompetent adenovirus 26 vector encoding the F protein stabilized in prefusion conformation. METHODS: This phase 1 clinical trial was performed in healthy adults aged ≥60 years. Seventy-two participants received 1 or 2 intramuscular injections of low-dose (LD; 5 × 1010 vector particles) or high-dose (HD; 1 × 1011 vector particles) Ad26.RSV.preF vaccine or placebo, with approximately 12 months between doses and 2-year follow-up for safety and immunogenicity outcomes. RESULTS: Solicited adverse events were reported by 44% of vaccine recipients and were transient and mild or moderate in intensity. No serious adverse events were related to vaccination. After the first vaccination, geometric mean titers for RSV-A2 neutralization increased from baseline (432 for LD and 512 for HD vaccine) to day 29 (1031 for LD and 1617 for HD). Pre-F-specific antibody geometric mean titers and median frequencies of F-specific interferon γ-secreting T cells also increased substantially from baseline. These immune responses were still maintained above baseline levels 2 years after immunization and could be boosted with a second immunization at 1 year. CONCLUSIONS: Ad26.RSV.preF (LD and HD) had an acceptable safety profile and elicited sustained humoral and cellular immune responses after a single immunization in older adults.

Asunto(s)

Adenoviridae , Vectores Genéticos , Infecciones por Virus Sincitial Respiratorio/prevención & control , Vacunas contra Virus Sincitial Respiratorio/inmunología , Virus Sincitial Respiratorio Humano/inmunología , Proteínas Virales de Fusión/inmunología , Adenoviridae/genética , Factores de Edad , Anciano , Anciano de 80 o más Años , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Femenino , Vectores Genéticos/genética , Humanos , Inmunidad Celular , Inmunogenicidad Vacunal , Masculino , Persona de Mediana Edad , Vacunas contra Virus Sincitial Respiratorio/efectos adversos , Vacunas contra Virus Sincitial Respiratorio/genética , Virus Sincitial Respiratorio Humano/genética , Vacunación , Proteínas Virales de Fusión/genética

RESUMEN

Small laboratory animals are powerful models for investigating in vivo viral pathogenesis of a number of viruses. For adenoviruses (AdVs), however, species-specificity poses limitations to studying human adenoviruses (HAdVs) in mice and other small laboratory animals. Thus, this review covers work on naturally occurring mouse AdVs, primarily mouse adenovirus type 1 (MAdV-1), a member of the species Murine mastadenovirus A. Molecular genetics, virus life cycle, cell and tissue tropism, interactions with the host immune response, persistence, and host genetics of susceptibility are described. A brief discussion of MAdV-2 (member of species Murine mastadenovirus B) and MAdV-3 (member of species Murine mastadenovirus C) is included. We report the use of MAdVs in the development of vectors and vaccines.

Asunto(s)

Infecciones por Adenoviridae/veterinaria , Mastadenovirus/patogenicidad , Animales , Regulación Viral de la Expresión Génica , Interacciones Huésped-Patógeno , Mastadenovirus/genética , Mastadenovirus/fisiología , Ratones , Especificidad de la Especie , Proteínas Virales/genética , Tropismo Viral

RESUMEN

Cigarette smoke (CS) is the primary risk factor for chronic obstructive pulmonary disease (COPD) and dampens antiviral response, which increases viral infections and leads to COPD acute exacerbation (AECOPD). Adenovirus, a nonenveloped DNA virus, is linked with AECOPD, whose DNAs trigger innate immune response via interacting with pattern recognition receptors (PRRs). Stimulator of interferon genes (STING), as a cytosolic DNA sensor, participates in adenovirus-induced interferon ß (IFNß)-dependent antiviral response. STING is involved in various pulmonary diseases, but role of STING in pathogenesis of AECOPD is not well documented. In the present study, we explored relationship between STING and AECOPD induced by recombinant adenovirus vectors (rAdVs) and CS in wild type (WT) and STING-/- mice; and also characterized the inhibition of STING- IFNß pathway in pulmonary epithelium exposed to cigarette smoke extract (CSE). We found that CS or CSE exposure alone dramatically inhibited STING expression, but not significantly effected IFNß production. Moreover, CS or CSE-exposed significantly suppressed activation of STING-IFNß pathway induced by rAdVs and suppressed clearance of rAdVs DNA. Inflammation, fibrosis and emphysema of lung tissues were exaggerated when treated with CS plus rAdVs, which further deteriorate in absences of STING. In A549 cells with knockdown of STING, we also observed enhancing apoptosis related to emphysema, especially CSE and adenovirus vectors in combination. Therefore, STING may play a protective role in preventing the progress of COPD.

Asunto(s)

Infecciones por Adenoviridae/genética , Vectores Genéticos/genética , Interferón beta/genética , Enfermedad Pulmonar Obstructiva Crónica/genética , Humo/efectos adversos , Productos de Tabaco/efectos adversos , Células A549 , Adenoviridae/efectos de los fármacos , Infecciones por Adenoviridae/tratamiento farmacológico , Animales , Línea Celular , Línea Celular Tumoral , Vectores Genéticos/efectos de los fármacos , Humanos , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/genética , Inflamación/tratamiento farmacológico , Inflamación/genética , Pulmón/efectos de los fármacos , Pulmón/virología , Ratones , Ratones Endogámicos C57BL , Enfermedad Pulmonar Obstructiva Crónica/virología , Enfisema Pulmonar/tratamiento farmacológico , Enfisema Pulmonar/genética , Enfisema Pulmonar/virología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Fumar/efectos adversos

RESUMEN

The aims of this study were to generate periodontal ligament (PDL) cells that have adenovirus- or lentivirus-mediated overexpression of human telomerase reverse transcriptase (hTERT) and to compare the osteogenic and proliferative abilities of the two cell lines to establish an efficient and stable cell model that will be more suitable for studies of PDL regeneration. After construction of the recombinant adenovirus plasmid pAd-pshuttle-cmv-hTERT, human PDL cells were infected by packaged adenovirus and lentivirus particles to establish two PDL cell lines. The expression levels of hTERT and mRNA for alkaline phosphatase, osteopontin, osteocalcin, bone sialoprotein, core-binding factor (runt-related transcription factor 2), and type I collagen were assessed for each cell line. After culture in osteoinductive culture medium for 14 days, the PDL cells were stained with alizarin red to observe formation of mineralized nodules, and proliferation activity was measured with a CCK-8 kit. A quantitative polymerase chain reaction assay indicated that the two transduced cell lines expressed hTERT levels that were significantly higher than that seen for normal PDL cells. Expression of all osteogenic genes tested, with the exception of osteopontin, was higher for both the adenovirus- and lentivirus-transduced cells relative to normal PDL cells. The expression of bone sialoprotein, osteocalcin, and runt-related transcription factor 2 in adenovirus-transduced cells was significantly higher than that for lentivirus-transduced cells. Alizarin red staining showed that the adenovirus-transduced cell line produced more mineralized nodules than the lentivirus-transduced cell line, whereas a CCK-8 test showed that the adenovirus-transduced cell line had higher proliferation activity than lentivirus-transduced cells. In conclusion, a PDL cell line established by adenovirus transduction had superior osteogenic differentiation and proliferative activity compared to the cell line produced by lentivirus transduction. The results indicate that PDL cells having adenovirus-mediated expression of hTERT would be a more suitable model for studies of PDL regeneration.

Asunto(s)

Adenoviridae/genética , Lentivirus/genética , Ligamento Periodontal/citología , Telomerasa/genética , Línea Celular , Proliferación Celular , Vectores Genéticos , Humanos , Osteogénesis/genética

RESUMEN

In situ sustained release of endogenous growth factors from cells is a challenge for repair and regeneration of tissue. Although recombinant adenovirus vectors are an effective delivery system that can prolong the release of growth factors and is very suitable for the therapy of growth factors, these recombinant adenovirus vectors that are widely used at present have low safety and stability in terms of long-term expression. In this study, the above problems are solved by knocking out both E1 and E3 genes at the same time and directly inserting the gene fragments encoding target proteins after the inverted terminal repeats. Finally, the combination of gene therapy with tissue engineering in regeneration and repair of full-thickness defects of osteochondral tissue are applied as an example. The results show that this strategy can achieve complete repair of articular osteochondral defects and recovery of their function, and meanwhile solve the problems of low safety and expression instability of recombinant adenovirus vectors. This method provides a bright prospect for the application of gene enhanced tissue engineering in the regeneration and repair of joint tissue, and also provides a reference for the repair and regeneration of other tissues.

Asunto(s)

Cartílago Articular , Condrogénesis , Terapia Genética/métodos , Regeneración , Ingeniería de Tejidos/métodos , Adenoviridae/genética , Animales , Proteína Morfogenética Ósea 2/genética , Cartílago Articular/metabolismo , Cartílago Articular/patología , Células Cultivadas , Condrogénesis/efectos de los fármacos , Condrogénesis/genética , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Conejos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Regeneración/efectos de los fármacos , Regeneración/genética , Andamios del Tejido , Proteína Wnt3A/genética

RESUMEN

Cytomegalovirus (CMV) and non-replicating adenoviral vectors can induce expanded, sustained effector-memory CD8+ T-cell responses, termed "memory inflation". During murine CMV (MCMV) infection, CD4+ Tcells maintain inflationary virus-specific CD8+ T-cell responses via IL-2 but not IL-21. Adenovirus vector vaccination can induce phenotypically, functionally and transcriptionally similar inflationary responses, but it is not known how IL-21 influences the inflating memory response to adenoviral vaccination. Here, we show that IL-21 is an absolute requirement for induction and maintenance of vaccine-derived inflationary CD8+ T-cell responses. These data indicate that the induction pathway of inflationary Ad-LacZ T-cells is distinct from inflationary MCMV-specific T-cells and is highly dependent on IL-21. Our observations highlight a fundamental difference in the mechanism by which adenovirus vectors and MCMV drive inflationary T-cell responses.

Asunto(s)

Vacunas contra el Adenovirus/administración & dosificación , Vacunas contra el Adenovirus/inmunología , Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica , Interleucinas/metabolismo , Animales , Interleucinas/genética , Ratones Endogámicos C57BL , Ratones Transgénicos

RESUMEN

Gene delivery vectors that do not rely on host cell genome integration offer several advantages for gene transfer, chiefly the avoidance of insertional mutagenesis and position effect variegation. However, unless engineered for replication and segregation, nonintegrating vectors will dilute progressively in proliferating cells, and are not exempt of epigenetic effects. This article provides an overview of the main nonintegrating viral (adenoviral, adeno-associated viral, integration-deficient retro-lentiviral, poxviral), and nonviral (plasmid vectors, artificial chromosomes) vectors used for preclinical and clinical cell and gene therapy applications. Particular emphasis is placed on their use in hematologic disease.

Asunto(s)

Terapia Genética , Vectores Genéticos/genética , Adenoviridae/genética , Animales , Ensayos Clínicos como Asunto/historia , Dependovirus/genética , Edición Génica , Expresión Génica , Técnicas de Transferencia de Gen , Terapia Genética/efectos adversos , Terapia Genética/historia , Terapia Genética/métodos , Vectores Genéticos/clasificación , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Plásmidos/genética , Poxviridae/genética , Transducción Genética

RESUMEN

Malaria is caused by parasites of the genus Plasmodium, which are transmitted to humans by the bites of Anopheles mosquitoes. After the elimination of Plasmodium falciparum, it is predicted that Plasmodium vivax will remain an important cause of morbidity and mortality outside Africa, stressing the importance of developing a vaccine against P. vivax malaria. In this study, we assessed the immunogenicity and protective efficacy of two P. vivax antigens, apical membrane antigen 1 (AMA1) and the 42-kDa C-terminal fragment of merozoite surface protein 1 (MSP142) in a plasmid recombinant DNA prime/adenoviral (Ad) vector boost regimen in Aotus monkeys. Groups of 4 to 5 monkeys were immunized with plasmid DNA alone, Ad alone, prime/boost regimens with each antigen, prime/boost regimens with both antigens, and empty vector controls and then subjected to blood-stage challenge. The heterologous immunization regimen with the antigen pair was more protective than either antigen alone or both antigens delivered with a single vaccine platform, on the basis of their ability to induce the longest prepatent period and the longest time to the peak level of parasitemia, the lowest peak and mean levels of parasitemia, the smallest area under the parasitemia curve, and the highest self-cure rate. Overall, prechallenge MSP142 antibody titers strongly correlated with a decreased parasite burden. Nevertheless, a significant proportion of immunized animals developed anemia. In conclusion, the P. vivax plasmid DNA/Ad serotype 5 vaccine encoding blood-stage parasite antigens AMA1 and MSP142 in a heterologous prime/boost immunization regimen provided significant protection against blood-stage challenge in Aotus monkeys, indicating the suitability of these antigens and this regimen for further development.

Asunto(s)

Antígenos de Protozoos/inmunología , Vacunas contra la Malaria/inmunología , Malaria Vivax/prevención & control , Proteínas de la Membrana/inmunología , Proteína 1 de Superficie de Merozoito/inmunología , Proteínas Protozoarias/inmunología , Vacunas de ADN/inmunología , Anemia/prevención & control , Animales , Anticuerpos Antiprotozoarios/sangre , Aotidae , Modelos Animales de Enfermedad , Femenino , Vacunas contra la Malaria/administración & dosificación , Malaria Vivax/inmunología , Masculino , Parasitemia/prevención & control , Resultado del Tratamiento , Vacunas de ADN/administración & dosificación